Mechanism of the Reaction of Peroxynitrite with Mn- Superoxide Dismutase: Nitration of Critical Tyrosine- 34

VERONICA DEMICHELI; DIEGO MORENO; GABRIEL E. JARA; SEBASTIAN CARBALLAL; CELIA QUIJANO; GERARDO FERRER-SUETA; NATALIA RIOS; DARIO ESTRIN; MARCELO MARTI; RAFAEL RADI

Congreso; Reunión Anual de la Society for Free Radical Biology and Medicine; 2014

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme located at the mitochondrial matrix that acts as a superoxide detoxifier. Superoxide, a free radical formed in high concentrations in this organelle can react with nitric oxide to lead to peroxynitrite formation. This reaction, that takes place at near diffusion limited rates, can compete with MnSOD for superoxide. Since MnSOD can be inactivated by peroxynitrite, this leads to a negative feedback that leads to the formation of more peroxynitrite in vivo. One of the most important bio-markers for peroxynitrite formation in vivo is tyrosine nitration, a post-translational modification that can alter both the structure and function of a protein. One of the most widely reported nitrated protein is MnSOD, which, when nitrated at specific tyrosine (Tyr34), leads to a complete inactivation. The reaction between MnSOD and peroxynitrite occurs via a direct reaction with a second order reaction constant of 1.0 x 105M-1s-1, according to reports by Quijano et al. Recently, a report by Surmeli et al, using the E. Coli MnSOD reported a constant with a value of several orders of magnitude inferior than the one previously reported and proposed a different mechanism of reaction. Given the controversy, is the aim of this study to settle the value of the second order reaction constant, using both direct and indirect spectroscopic methods. The results obtained showed a value of 1.92 x 104M-1s-1, which is in accordance with a metal-catalyzed reaction, involving the oxidation of the metal center, according to the following equation: E-Mn3+ + ONOO-  E-Mn4+=O + NO2.. In order to clarify the mechanism proposed by the different groups, theoretical methods (QM/MM) were also used, which allowed us the study, at a molecular level, of different approaches to the reaction between MnSOD and peroxynitrite, focusing on the active site of the enzyme. The results obtained by both experimental and theoretical measurements are consistent with a metal-catalyzed reaction that involves the formation of a Mn4+=O, which explains the site-specific nitration of Tyr34 located 5Å away from the metal, a distance close enough for the metal to attack this critical nearby residue.